Tadpole tail regeneration in Xenopus
Biochemical Society Transactions
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Yaoyao Chen*†, Nick R. Love*‡ and Enrique Amaya*1 *The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K. †Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, U.K. ‡RIKEN Center for Developmental Biology, 650-0047 Kobe, Japan
Abstract Some organisms have a remarkable ability to heal wounds without scars and to regenerate complex tissues following injury. By gaining a more complete understanding of the biological mechanisms that promote scar-free healing and tissue regeneration, it is hoped that novel treatments that can enhance the healing and regenerative capacity of human patients can be found. In the present article, we briefly examine the genetic, molecular and cellular mechanisms underlying the regeneration of the Xenopus tadpole tail.
Introduction Although mammals have relatively poor capacities to heal wounds and regenerate complex tissues, many organisms across the animal kingdom have remarkably good abilities to regenerate body parts following traumatic injuries. In these cases, the regeneration programme must be very robust, as no two injuries are ever the same. Successful regeneration relies on a co-ordinated orchestration of numerous processes, such as gene activation, signalling, cell proliferation, morphogenesis, tissue patterning and differentiation. How these proceed in a synchronized way during regeneration (and cease when regeneration is finished) is not very well understood. Ultimately, it is hoped that a better understanding of these aspects of regeneration will facilitate the development of improved and more efficient healing and regenerative therapies following traumatic injuries [1]. Investigations using invertebrate and vertebrate model organisms have greatly contributed to our understanding of the biology of regeneration [2,3]. Among vertebrate organisms, urodele amphibians are of particular note, as they have been reported to completely regenerate their arms, legs, tails, eye lens and jaw portions following removal [4,5]. In the present review, we examine another well-studied model of vertebrate regeneration: the anuran amphibian Xenopus, which is capable of fully regenerating its tail Key words: metabolism, reactive oxygen species, tissue regeneration, wound healing, Xenopus. Abbreviations: FGF, fibroblast growth factor; HIF, hypoxia-inducible factor; Ldha, lactate dehydrogenase A; Nrx, nucleoredoxin; Pdk1, pyruvate dehydrogenase kinase 1; PEP, phosphoenolpyruvate; PK, pyruvate kinase; ROS, reactive oxygen species; Slc, solute carrier. 1
To whom correspondence manchester.ac.uk).
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Biochem. Soc. Trans. (2014) 42, 617–623; doi:10.1042/BST20140061
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following amputation throughout its larval stage. For at least 60 years, scientists have examined the regeneration of the Xenopus tadpole tail [6–9], and thus the literature pertaining to this model has been reviewed previously [10–12]. In the present review, we briefly examine the genetic, molecular and cellular mechanisms underlying the regeneration of the Xenopus tadpole tail before focusing on recent evidence concerning the metabolic regulation of the regenerative process.
The Xenopus tadpole tail regeneration model
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As an experimental model, aquatic Xenopus frogs have numerous advantages [13]: they are relatively inexpensive to house; tadpoles can be generated in very large numbers, by either induction of natural matings, following the administration of hormones, or by in vitro fertilizations at any time of the year; the Xenopus model has extensive genomic resources, including the published genome of Xenopus tropicalis [14] and there exist over a million ESTs and thousands of gene clones, which expedite gainof-function and in situ hybridization studies [15–17]. Moreover, Xenopus transgenic technology permits tissuespecific, inducible and binary transgene expression systems that facilitate tail-regeneration experiments [18–21]. Finally, the recent development of genome-editing techniques will greatly enhance our ability to study the genetic basis of regeneration in this system [22]. The tadpole tail is a complex appendage, containing many axial tissues that are also found in humans (e.g. the spinal cord C The
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and dorsal aorta). Moreover, the Xenopus tadpole tail contains a notochord, a dense network of neurons, skeletal and smooth muscle, lymphatic vasculature, and an epidermis dotted with black pigmented melanophores. All of these tissues regenerate and return to a fully functional tail appendage within 1 week of amputation.
The early, intermediate and late phases of Xenopus tadpole tail regeneration Tadpole tail regeneration proceeds through three phases: early, intermediate and late [23,24] (Figure 1). In the early phase (within the first 6 h of amputation), the site of injury undergoes scarless wound healing. This stage is also associated with an acute inflammatory response [24,25], which can be assayed using the histological stain Sudan Black B, which strongly reacts with neutrophil granules in inflammatory cells, [26] or by in situ hybridization using marker genes, such as Mmp7 (matrix metalloproteinase 7) or Mpo (myeloperoxidase) [27,28]. The inflammatory response can also be imaged using time-lapse microscopy on transgenic lines, which label myeloid cells with GFP [29,30]. At the injury site, inflammatory cells help to prevent microbial infection and participate in other phagocytic and nonphagocytic duties. Several other activities have been shown to be critical during the early phase of tail regeneration. For example, during the first 6 h of tail amputation, the V-ATPase (vacuolar ATPase) H + pump is activated and results in an electrically polarized injured tail [31], which is required for proper regeneration. In addition, during this phase of regeneration, a moderate number of cells at the injured site undergo apoptosis, and this is required for tail regeneration to proceed normally [32]. Given the evidence that amputation-induced apoptotic cells can release growth factors during Hydra regeneration [33], it is possible that these apoptotic cells at the distal part of the injured tail are also a source of proregenerative signals during Xenopus tail regeneration. The next phase of regeneration, the intermediate phase (∼24–36 h after amputation), is classified by the formation of regenerative tissue distal to the wound site. Although this nascent regenerative tissue is sometimes referred to as the ‘blastema’, we (and others) prefer to refer to this tissue as the ‘regenerative bud’ [34]. The regenerative bud is highly dynamic and can take various shapes during regeneration. Although the exact role of the regenerative bud is not entirely understood, its formation is generally thought to be required for tail regeneration. In addition, grafting experiments have shown that the cells of the regenerative bud do not appear to contribute to or ‘seed’ the regenerating muscle, spinal cord or notochord tissues [35]. However, the regenerative bud clearly possesses an intrinsic patterning ability and can promote tissue outgrowth: transplanting a genetically labelled regenerative bud into the head region of a tadpole will cause an ectopic tail to grow, and the ectopic tail’s epidermis retains the genetic label [36]. C The
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Following the formation of the regenerative bud, the overt regrowth of the tail appendage begins during the late phase of tadpole tail regeneration. During this time, the vascular, neuronal, notochord and muscle tissues all repopulate the new tail from ∼24–36 h after amputation until ∼5–9 days after amputation [30]. As one might expect, successful tail regeneration requires the activity of many growth factors, including the BMP (bone morphogenetic protein), Notch, Wnt, FGF (fibroblast growth factor) and TGFβ (transforming growth factor β) [20,37,38]. Given the interest in spinal cord regeneration following human injury, the mechanisms of tadpole spinal cord regeneration are particularly intriguing. Grafting experiments using GFP-expressing transgenic tadpoles have elegantly shown that the injured spinal cord (and notochord) provides the cellular source for their regenerative portions [35]. The presence of the spinal cord is essential for regeneration, as its removal before amputation will severely compromise the regenerative response [39]. Tail muscle regenerates from a reservoir of resident muscle stem cells, called satellite cells [35]. It is now appreciated that the transcription factor Pax7 is essential for satellite cells to be able to mount a regenerative response [40]. More recent studies have used fluorophores to track the dynamic behaviour of myofibrils during tail muscle regeneration, suggesting that injured tail muscle fibres show some signs of dedifferentiation following amputation [41]. The regeneration of the tadpole tail vasculature has only been minimally studied [24]. However, there currently exist several transgenic lines in both Xenopus laevis [42] and X. tropicalis [24] that can be used to image the regeneration of the vasculature in vivo [30]. The availability of these lines should facilitate the study of tadpole vascular regeneration. It is expected that the vasculature is essential for providing oxygen and nourishment to the regenerating tail.
Gene expression during tail regeneration Tail amputation and the subsequent regeneration programme result in the alteration of gene expression level in thousands of genes during regeneration [23,24]. The change in gene expression following amputation is likely to depend on remodelling of the epigenetic landscape of the genome, and, consistent with this prediction, HDAC (histone deacetylase) activity has recently been shown to be essential for amputation-induced change in mRNA transcription [34,43]. One fundamental question regarding tail regeneration is whether the regeneration gene expression programme is similar to that seen during the initial tail developmental programme. This question of ‘recapitulation’ was addressed by Mochii and colleagues, who found that several developmental genes (i.e. chordin, noggin, Xshh and delta1) were expressed during development but not during regeneration [44]. Moreover, in a separate study, it was also found that the expression of abdominal B-type Hox genes hoxc10, hoxa13 and hoxd13 were not the same during tail embryonic development and regeneration [45].
Biochemical Society Annual Symposium No. 81: Biochemical Determinants of Tissue Regeneration
Figure 1 Overview of events that occur during tail regeneration Tail regeneration proceeds through three phases. Soon after injury, the amputation site begins to produce ROS, and this production is sustained through the regeneration process. During regeneration, a large cohort of genes change in expression and many of these are associated with signalling and metabolism (depicted in the close-up inset from the 24 h after amputation time point). Many of these changes would be predicted to lead to increased anabolic pathways and attenuation of catabolic pathways, thereby facilitating tissue growth and regeneration. G6P, glucose 6-phosphate; g6pd, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway.
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However, other genes, such as Fgf20, clearly reinitiate their developmental expression patterns during tail regeneration [17,38]. Thus regeneration recapitulates some gene expression patterns exhibited during tail development, but not all genes do so.
The role of reactive oxygen species during tail regeneration We performed a microarray screen aimed at examining the changes in gene expression during Xenopus tail regeneration, uncovering a number of co-ordinately up-regulated genes involved in the production of ROS (reactive oxygen species) such as H2 O2 [24]. Indeed, by using an oxidativesensitive ratiometric reporter fluorophore (HyPerYFP), we were able to confirm that tail amputation and the subsequent regenerative response was associated with a significant increase in the production of intracellular ROS levels [46] (Figure 1). By monitoring the entire process of tail regeneration, we found that this oxidative change, which is equivalent to 50–200 μM H2 O2 , lasts for at least several days before it eventually wanes. Interestingly, this amputation-induced ROS not only precedes the recruitment of inflammatory cells, but also functions independently of inflammatory cells. H2 O2 and other ROS, originally thought to cause stress and apoptosis, now have been increasingly appreciated as important regulators of various cellular processes, including cell metabolism, motility, proliferation and signalling [47,48]. Indeed, we found that decreasing ROS levels during tail regeneration via chemical inhibitors or gene-knockdown strategies, especially during the first 72 h after amputation, resulted in sluggish cell proliferation and impaired tail regeneration [46]. To better understand the potential mechanisms that might be affected by ROS during the process of tail regeneration, we asked whether Wnt/β-catenin and/or FGF signalling acted downstream of ROS production, as both of these growth factors had been shown previously to be critical for tail regeneration [38]. By using a X. tropicalis Wnt/β-catenin reporter line [49], we observed a sustained activation of Wnt/β-catenin signalling from 24 h after amputation and we found that inhibiting ROS using chemical compounds resulted in a significant decrease in the activation of Wnt/βcatenin signalling. It has been reported that Wnt/β-catenin signalling can be modulated by H2 O2 through a small redox-sensitive protein, Nrx (nucleoredoxin) [50]. Further investigation is needed to assess whether Nrx also mediates the changes in Wnt/β-catenin signalling activity caused by increased ROS levels during tail regeneration. FGF signalling has also been shown to act downstream of Wnt/β-catenin signalling during vertebrate appendage regeneration, as its inhibition blocks proliferation of blastemal cells during zebrafish caudal fin regeneration [51]. Among all of the genes that encode FGF ligands, we and others found that Fgf20 was among the most highly upregulated Fgf genes found during both X. laevis and X. tropicalis tail regeneration [24,38]. We and others also found C The
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that inhibiting the expression of Fgf20 or its activity by either chemical or genetic means results in impaired tail regeneration [38,46]. Notably, the activation of Fgf20 gene expression during tail regeneration was significantly impaired when we treated tadpoles with ROS inhibitors, suggesting that the production of ROS following tail amputation is important for proper FGF signalling during regeneration [46]. Taken together, we speculate that the amputationinduced ROS facilitates the activation of Wnt/β-catenin and FGF signalling, both of which are crucial for tadpole tail regeneration. However, apart from modulating these two signalling pathways, ROS also affect other aspects of cellular physiology, such as cellular metabolism, thereby potentially creating a permissive environment for tissues to repair and regenerate.
Towards a greater understanding of the regulation and role of cellular metabolism during regeneration The metabolism of a living homoeostatic organism is well balanced between catabolic and anabolic reactions. However, under circumstances that require tissue regrowth, a higher rate of anabolism is intuitively preferred to meet the demands of new cellular components. In fact, scientists noticed decades ago that the citric acid cycle was not favoured during preblastemic and blastemic phases of tissue regeneration, and that glucose metabolism was tuned towards glycolysis and the PPP (pentose phosphate pathway) to provide intermediate metabolites to fuel anabolic processes, as well as to create an acidic local environment which was thought to be essential for many processes of regeneration [52]. However, it has remained unclear exactly how glucose metabolism is modulated during regeneration, since the earlier studies were restrained by the lack of decoded genome information and large-scale ‘-omic’ approaches. However, now with the published genome resources of X. tropicalis [14] and X. laevis, our genome-wide microarray analysis of X. tropicalis tail regeneration, as well as other large-scale genetic analysis of Xenopus tissue repair [23,53–55], we are better able to incorporate these important genetic tools to gain more insight into our understanding of the regulation and role of metabolism during tissue regeneration. We found that the expression of a large number of metabolic genes was greatly altered during the early, intermediate and late phases of tail regeneration [24] (Figure 1). When we specifically looked at genes related to glucose metabolism, we found that the expression of genes that stimulate glucose intake, such as those encoding leptin and proinsulin, and genes encoding subunits of glucose transporters, such as Slc2a3 (solute carrier 2 A3), were significantly induced after tail amputation. These data strongly suggest that regenerating tadpole tails actively increase glucose consumption. Meanwhile, our gene analysis also revealed that mRNA levels of Hif1a (hypoxia-inducible factor 1α), a gene known to reduce mitochondrial activity [56], was increased during
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tail regeneration [24]. Moreover, it is likely that the HIF1α protein is also stabilized at the post-translational level as amputation cuts down the blood supply to the injured area and creates a hypoxic environment {a condition which abrogates the prolyl hydroxylation of HIF1α and allows it to escape recognition by the pVHL (von Hippel–Lindau protein) ubiquitin ligase complex for degradation [57]}. Notably, intracellular ROS have also been reported to inhibit the catalytic activity of the prolyl hydroxylase domain and therefore negatively regulate HIF1α degradation [58– 60]. As a result, HIF1α accumulates in the nucleus and transactivates HIF-responsive genes, including many key metabolic genes, such as Pdk1, Ldha and Slc2a1 [56,61– 64]. These metabolic genes may have important roles in shaping the metabolic environment during tissue repair: Pdk1 encodes pyruvate dehydrogenase kinase 1 which inhibits the conversion of pyruvate into acetyl-CoA by inactivating pyruvate dehydrogenase; Ldha encodes lactate dehydrogenase A which catalyses the conversion of pyruvate into lactate; and Slc2a1 encodes a major glucose transporter, GLUT1, which can further enhance the glucose intake. Thus these genes may act synergistically to promote glycolysis and restrain carbohydrates from entering the tricarboxylic acid cycle [65–67]. Another important checkpoint that controls the balance between glycolysis and oxidative phosphorylation is the conversion of PEP (phosphoenolpyruvate) into pyruvate. The enzyme which regulates this rate-limiting step has two splice isoforms, PK (pyruvate kinase) M1 and M2 [68]. Although it is yet to be tested which isoform is expressed during tadpole tail regeneration, it was suggested that the expression of PKM2 can be induced by HIF1α through a positive-feedback loop [69], therefore PKM2 may indeed be the major form of PKMs expressed in the hypoxic tail. Unlike PKM1, which is constitutively active, PKM2 can switch between an active tetrameric and an inactive dimeric form [70], and is susceptible to inhibition by growth factor signalling and ROS oxidation [71,72]. Thus we speculate that the PKM2-mediated inefficient conversion of PEP also facilitates the metabolic shift towards glycolysis during tail regrowth. Altogether, changes in gene expression, growth factor signalling and ROS co-operate to reprogramme metabolism during tail regeneration to meet the synthetic demands of building new biomass during appendage regeneration. A more complete understanding of tissue regeneration will include insights into whether modulating metabolic programming may improve tissue regeneration outcomes in other animal models that are less competent to regenerate, such as mammals.
Funding This work was supported by the Wellcome Trust [grant number WT082450MA], the Healing Foundation and the National Science Foundation.
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Received 14 March 2014 doi:10.1042/BST20140061
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Yaoyao Chen*†, Nick R. Love*‡ and Enrique Amaya*1 *The Healing Foundation Centre, Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, U.K. †Wellcome Trust–Medical Research Council Cambridge Stem Cell Institute, Tennis Court Road, Cambridge CB2 1QR, U.K. ‡RIKEN Center for Developmental Biology, 650-0047 Kobe, Japan
Abstract Some organisms have a remarkable ability to heal wounds without scars and to regenerate complex tissues following injury. By gaining a more complete understanding of the biological mechanisms that promote scar-free healing and tissue regeneration, it is hoped that novel treatments that can enhance the healing and regenerative capacity of human patients can be found. In the present article, we briefly examine the genetic, molecular and cellular mechanisms underlying the regeneration of the Xenopus tadpole tail.
Introduction Although mammals have relatively poor capacities to heal wounds and regenerate complex tissues, many organisms across the animal kingdom have remarkably good abilities to regenerate body parts following traumatic injuries. In these cases, the regeneration programme must be very robust, as no two injuries are ever the same. Successful regeneration relies on a co-ordinated orchestration of numerous processes, such as gene activation, signalling, cell proliferation, morphogenesis, tissue patterning and differentiation. How these proceed in a synchronized way during regeneration (and cease when regeneration is finished) is not very well understood. Ultimately, it is hoped that a better understanding of these aspects of regeneration will facilitate the development of improved and more efficient healing and regenerative therapies following traumatic injuries [1]. Investigations using invertebrate and vertebrate model organisms have greatly contributed to our understanding of the biology of regeneration [2,3]. Among vertebrate organisms, urodele amphibians are of particular note, as they have been reported to completely regenerate their arms, legs, tails, eye lens and jaw portions following removal [4,5]. In the present review, we examine another well-studied model of vertebrate regeneration: the anuran amphibian Xenopus, which is capable of fully regenerating its tail Key words: metabolism, reactive oxygen species, tissue regeneration, wound healing, Xenopus. Abbreviations: FGF, fibroblast growth factor; HIF, hypoxia-inducible factor; Ldha, lactate dehydrogenase A; Nrx, nucleoredoxin; Pdk1, pyruvate dehydrogenase kinase 1; PEP, phosphoenolpyruvate; PK, pyruvate kinase; ROS, reactive oxygen species; Slc, solute carrier. 1
To whom correspondence manchester.ac.uk).
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following amputation throughout its larval stage. For at least 60 years, scientists have examined the regeneration of the Xenopus tadpole tail [6–9], and thus the literature pertaining to this model has been reviewed previously [10–12]. In the present review, we briefly examine the genetic, molecular and cellular mechanisms underlying the regeneration of the Xenopus tadpole tail before focusing on recent evidence concerning the metabolic regulation of the regenerative process.
The Xenopus tadpole tail regeneration model
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As an experimental model, aquatic Xenopus frogs have numerous advantages [13]: they are relatively inexpensive to house; tadpoles can be generated in very large numbers, by either induction of natural matings, following the administration of hormones, or by in vitro fertilizations at any time of the year; the Xenopus model has extensive genomic resources, including the published genome of Xenopus tropicalis [14] and there exist over a million ESTs and thousands of gene clones, which expedite gainof-function and in situ hybridization studies [15–17]. Moreover, Xenopus transgenic technology permits tissuespecific, inducible and binary transgene expression systems that facilitate tail-regeneration experiments [18–21]. Finally, the recent development of genome-editing techniques will greatly enhance our ability to study the genetic basis of regeneration in this system [22]. The tadpole tail is a complex appendage, containing many axial tissues that are also found in humans (e.g. the spinal cord C The
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and dorsal aorta). Moreover, the Xenopus tadpole tail contains a notochord, a dense network of neurons, skeletal and smooth muscle, lymphatic vasculature, and an epidermis dotted with black pigmented melanophores. All of these tissues regenerate and return to a fully functional tail appendage within 1 week of amputation.
The early, intermediate and late phases of Xenopus tadpole tail regeneration Tadpole tail regeneration proceeds through three phases: early, intermediate and late [23,24] (Figure 1). In the early phase (within the first 6 h of amputation), the site of injury undergoes scarless wound healing. This stage is also associated with an acute inflammatory response [24,25], which can be assayed using the histological stain Sudan Black B, which strongly reacts with neutrophil granules in inflammatory cells, [26] or by in situ hybridization using marker genes, such as Mmp7 (matrix metalloproteinase 7) or Mpo (myeloperoxidase) [27,28]. The inflammatory response can also be imaged using time-lapse microscopy on transgenic lines, which label myeloid cells with GFP [29,30]. At the injury site, inflammatory cells help to prevent microbial infection and participate in other phagocytic and nonphagocytic duties. Several other activities have been shown to be critical during the early phase of tail regeneration. For example, during the first 6 h of tail amputation, the V-ATPase (vacuolar ATPase) H + pump is activated and results in an electrically polarized injured tail [31], which is required for proper regeneration. In addition, during this phase of regeneration, a moderate number of cells at the injured site undergo apoptosis, and this is required for tail regeneration to proceed normally [32]. Given the evidence that amputation-induced apoptotic cells can release growth factors during Hydra regeneration [33], it is possible that these apoptotic cells at the distal part of the injured tail are also a source of proregenerative signals during Xenopus tail regeneration. The next phase of regeneration, the intermediate phase (∼24–36 h after amputation), is classified by the formation of regenerative tissue distal to the wound site. Although this nascent regenerative tissue is sometimes referred to as the ‘blastema’, we (and others) prefer to refer to this tissue as the ‘regenerative bud’ [34]. The regenerative bud is highly dynamic and can take various shapes during regeneration. Although the exact role of the regenerative bud is not entirely understood, its formation is generally thought to be required for tail regeneration. In addition, grafting experiments have shown that the cells of the regenerative bud do not appear to contribute to or ‘seed’ the regenerating muscle, spinal cord or notochord tissues [35]. However, the regenerative bud clearly possesses an intrinsic patterning ability and can promote tissue outgrowth: transplanting a genetically labelled regenerative bud into the head region of a tadpole will cause an ectopic tail to grow, and the ectopic tail’s epidermis retains the genetic label [36]. C The
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Following the formation of the regenerative bud, the overt regrowth of the tail appendage begins during the late phase of tadpole tail regeneration. During this time, the vascular, neuronal, notochord and muscle tissues all repopulate the new tail from ∼24–36 h after amputation until ∼5–9 days after amputation [30]. As one might expect, successful tail regeneration requires the activity of many growth factors, including the BMP (bone morphogenetic protein), Notch, Wnt, FGF (fibroblast growth factor) and TGFβ (transforming growth factor β) [20,37,38]. Given the interest in spinal cord regeneration following human injury, the mechanisms of tadpole spinal cord regeneration are particularly intriguing. Grafting experiments using GFP-expressing transgenic tadpoles have elegantly shown that the injured spinal cord (and notochord) provides the cellular source for their regenerative portions [35]. The presence of the spinal cord is essential for regeneration, as its removal before amputation will severely compromise the regenerative response [39]. Tail muscle regenerates from a reservoir of resident muscle stem cells, called satellite cells [35]. It is now appreciated that the transcription factor Pax7 is essential for satellite cells to be able to mount a regenerative response [40]. More recent studies have used fluorophores to track the dynamic behaviour of myofibrils during tail muscle regeneration, suggesting that injured tail muscle fibres show some signs of dedifferentiation following amputation [41]. The regeneration of the tadpole tail vasculature has only been minimally studied [24]. However, there currently exist several transgenic lines in both Xenopus laevis [42] and X. tropicalis [24] that can be used to image the regeneration of the vasculature in vivo [30]. The availability of these lines should facilitate the study of tadpole vascular regeneration. It is expected that the vasculature is essential for providing oxygen and nourishment to the regenerating tail.
Gene expression during tail regeneration Tail amputation and the subsequent regeneration programme result in the alteration of gene expression level in thousands of genes during regeneration [23,24]. The change in gene expression following amputation is likely to depend on remodelling of the epigenetic landscape of the genome, and, consistent with this prediction, HDAC (histone deacetylase) activity has recently been shown to be essential for amputation-induced change in mRNA transcription [34,43]. One fundamental question regarding tail regeneration is whether the regeneration gene expression programme is similar to that seen during the initial tail developmental programme. This question of ‘recapitulation’ was addressed by Mochii and colleagues, who found that several developmental genes (i.e. chordin, noggin, Xshh and delta1) were expressed during development but not during regeneration [44]. Moreover, in a separate study, it was also found that the expression of abdominal B-type Hox genes hoxc10, hoxa13 and hoxd13 were not the same during tail embryonic development and regeneration [45].
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Figure 1 Overview of events that occur during tail regeneration Tail regeneration proceeds through three phases. Soon after injury, the amputation site begins to produce ROS, and this production is sustained through the regeneration process. During regeneration, a large cohort of genes change in expression and many of these are associated with signalling and metabolism (depicted in the close-up inset from the 24 h after amputation time point). Many of these changes would be predicted to lead to increased anabolic pathways and attenuation of catabolic pathways, thereby facilitating tissue growth and regeneration. G6P, glucose 6-phosphate; g6pd, glucose-6-phosphate dehydrogenase; PPP, pentose phosphate pathway.
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However, other genes, such as Fgf20, clearly reinitiate their developmental expression patterns during tail regeneration [17,38]. Thus regeneration recapitulates some gene expression patterns exhibited during tail development, but not all genes do so.
The role of reactive oxygen species during tail regeneration We performed a microarray screen aimed at examining the changes in gene expression during Xenopus tail regeneration, uncovering a number of co-ordinately up-regulated genes involved in the production of ROS (reactive oxygen species) such as H2 O2 [24]. Indeed, by using an oxidativesensitive ratiometric reporter fluorophore (HyPerYFP), we were able to confirm that tail amputation and the subsequent regenerative response was associated with a significant increase in the production of intracellular ROS levels [46] (Figure 1). By monitoring the entire process of tail regeneration, we found that this oxidative change, which is equivalent to 50–200 μM H2 O2 , lasts for at least several days before it eventually wanes. Interestingly, this amputation-induced ROS not only precedes the recruitment of inflammatory cells, but also functions independently of inflammatory cells. H2 O2 and other ROS, originally thought to cause stress and apoptosis, now have been increasingly appreciated as important regulators of various cellular processes, including cell metabolism, motility, proliferation and signalling [47,48]. Indeed, we found that decreasing ROS levels during tail regeneration via chemical inhibitors or gene-knockdown strategies, especially during the first 72 h after amputation, resulted in sluggish cell proliferation and impaired tail regeneration [46]. To better understand the potential mechanisms that might be affected by ROS during the process of tail regeneration, we asked whether Wnt/β-catenin and/or FGF signalling acted downstream of ROS production, as both of these growth factors had been shown previously to be critical for tail regeneration [38]. By using a X. tropicalis Wnt/β-catenin reporter line [49], we observed a sustained activation of Wnt/β-catenin signalling from 24 h after amputation and we found that inhibiting ROS using chemical compounds resulted in a significant decrease in the activation of Wnt/βcatenin signalling. It has been reported that Wnt/β-catenin signalling can be modulated by H2 O2 through a small redox-sensitive protein, Nrx (nucleoredoxin) [50]. Further investigation is needed to assess whether Nrx also mediates the changes in Wnt/β-catenin signalling activity caused by increased ROS levels during tail regeneration. FGF signalling has also been shown to act downstream of Wnt/β-catenin signalling during vertebrate appendage regeneration, as its inhibition blocks proliferation of blastemal cells during zebrafish caudal fin regeneration [51]. Among all of the genes that encode FGF ligands, we and others found that Fgf20 was among the most highly upregulated Fgf genes found during both X. laevis and X. tropicalis tail regeneration [24,38]. We and others also found C The
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that inhibiting the expression of Fgf20 or its activity by either chemical or genetic means results in impaired tail regeneration [38,46]. Notably, the activation of Fgf20 gene expression during tail regeneration was significantly impaired when we treated tadpoles with ROS inhibitors, suggesting that the production of ROS following tail amputation is important for proper FGF signalling during regeneration [46]. Taken together, we speculate that the amputationinduced ROS facilitates the activation of Wnt/β-catenin and FGF signalling, both of which are crucial for tadpole tail regeneration. However, apart from modulating these two signalling pathways, ROS also affect other aspects of cellular physiology, such as cellular metabolism, thereby potentially creating a permissive environment for tissues to repair and regenerate.
Towards a greater understanding of the regulation and role of cellular metabolism during regeneration The metabolism of a living homoeostatic organism is well balanced between catabolic and anabolic reactions. However, under circumstances that require tissue regrowth, a higher rate of anabolism is intuitively preferred to meet the demands of new cellular components. In fact, scientists noticed decades ago that the citric acid cycle was not favoured during preblastemic and blastemic phases of tissue regeneration, and that glucose metabolism was tuned towards glycolysis and the PPP (pentose phosphate pathway) to provide intermediate metabolites to fuel anabolic processes, as well as to create an acidic local environment which was thought to be essential for many processes of regeneration [52]. However, it has remained unclear exactly how glucose metabolism is modulated during regeneration, since the earlier studies were restrained by the lack of decoded genome information and large-scale ‘-omic’ approaches. However, now with the published genome resources of X. tropicalis [14] and X. laevis, our genome-wide microarray analysis of X. tropicalis tail regeneration, as well as other large-scale genetic analysis of Xenopus tissue repair [23,53–55], we are better able to incorporate these important genetic tools to gain more insight into our understanding of the regulation and role of metabolism during tissue regeneration. We found that the expression of a large number of metabolic genes was greatly altered during the early, intermediate and late phases of tail regeneration [24] (Figure 1). When we specifically looked at genes related to glucose metabolism, we found that the expression of genes that stimulate glucose intake, such as those encoding leptin and proinsulin, and genes encoding subunits of glucose transporters, such as Slc2a3 (solute carrier 2 A3), were significantly induced after tail amputation. These data strongly suggest that regenerating tadpole tails actively increase glucose consumption. Meanwhile, our gene analysis also revealed that mRNA levels of Hif1a (hypoxia-inducible factor 1α), a gene known to reduce mitochondrial activity [56], was increased during
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tail regeneration [24]. Moreover, it is likely that the HIF1α protein is also stabilized at the post-translational level as amputation cuts down the blood supply to the injured area and creates a hypoxic environment {a condition which abrogates the prolyl hydroxylation of HIF1α and allows it to escape recognition by the pVHL (von Hippel–Lindau protein) ubiquitin ligase complex for degradation [57]}. Notably, intracellular ROS have also been reported to inhibit the catalytic activity of the prolyl hydroxylase domain and therefore negatively regulate HIF1α degradation [58– 60]. As a result, HIF1α accumulates in the nucleus and transactivates HIF-responsive genes, including many key metabolic genes, such as Pdk1, Ldha and Slc2a1 [56,61– 64]. These metabolic genes may have important roles in shaping the metabolic environment during tissue repair: Pdk1 encodes pyruvate dehydrogenase kinase 1 which inhibits the conversion of pyruvate into acetyl-CoA by inactivating pyruvate dehydrogenase; Ldha encodes lactate dehydrogenase A which catalyses the conversion of pyruvate into lactate; and Slc2a1 encodes a major glucose transporter, GLUT1, which can further enhance the glucose intake. Thus these genes may act synergistically to promote glycolysis and restrain carbohydrates from entering the tricarboxylic acid cycle [65–67]. Another important checkpoint that controls the balance between glycolysis and oxidative phosphorylation is the conversion of PEP (phosphoenolpyruvate) into pyruvate. The enzyme which regulates this rate-limiting step has two splice isoforms, PK (pyruvate kinase) M1 and M2 [68]. Although it is yet to be tested which isoform is expressed during tadpole tail regeneration, it was suggested that the expression of PKM2 can be induced by HIF1α through a positive-feedback loop [69], therefore PKM2 may indeed be the major form of PKMs expressed in the hypoxic tail. Unlike PKM1, which is constitutively active, PKM2 can switch between an active tetrameric and an inactive dimeric form [70], and is susceptible to inhibition by growth factor signalling and ROS oxidation [71,72]. Thus we speculate that the PKM2-mediated inefficient conversion of PEP also facilitates the metabolic shift towards glycolysis during tail regrowth. Altogether, changes in gene expression, growth factor signalling and ROS co-operate to reprogramme metabolism during tail regeneration to meet the synthetic demands of building new biomass during appendage regeneration. A more complete understanding of tissue regeneration will include insights into whether modulating metabolic programming may improve tissue regeneration outcomes in other animal models that are less competent to regenerate, such as mammals.
Funding This work was supported by the Wellcome Trust [grant number WT082450MA], the Healing Foundation and the National Science Foundation.
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Received 14 March 2014 doi:10.1042/BST20140061
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